CN100372511C - Acellular dermal matrix and its preparing method - Google Patents
Acellular dermal matrix and its preparing method Download PDFInfo
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Abstract
The present invention discloses an acellular dermal matrix and a preparation method thereof. The acellular dermal matrix is a product which is obtained after the skin of a mammal is orderly processed by a sodium hydroxide solution and a detergent. The preparation method comprises the following steps: 1) the skin of the body surface of a mammal is stripped; 2) subcutaneous attached components are removed; 3) the skin is cleaned; 4) the skin is processed for 10 to 60 minutes by 2 to 5N of sodium hydroxide solution; 5) 2 to 5N of novel sodium hydroxide solution is replaced, and the skin is processed for 10 to 60 minutes; 6) the step (5) is repeated for 1 to 3 times; 6) the skin which is processed by the sodium hydroxide solution is processed by the detergent whose mass percentage concentration of 1 to 5 %; 8) the skin is cleaned by deheating raw water, and the acellular dermal matrix is obtained. The present invention has high practical application value in a medical field.
Description
Technical field
The present invention relates to acellular dermal matrix and preparation method thereof.
Background technology
Very long course has been passed through in the research of acellular dermal matrix, as far back as 1913, Loewe has just confirmed that dermal tissue can strengthen toughness, the growth of inhibition granulation tissue and the cicatrization of transplanting skin, reduce wound surface contracture degree, people begin to notice the important function of dermal tissue in skin transplantation thus; Heck in 1985 etc. report is used allosome corium as the transplanting carrier from the body surface skin, with the two cograft in animal model and fire victim cut the crust wound surface, obtained wound repair preferably and suppressed the effect of scar hyperplasia.But application after this and discovering, the immunological rejection that allogeneic skin graft took place mainly comes from cells such as epidermis cell, endotheliocyte and fibroblast, promoted thus corium acellularization research (Chen Bin etc. the progress of acellular dermal matrix. Chinese aesthetic medicine 2001,10 (4): 358-359.).Nineteen ninety-five, Livesey etc. at first report and make acellular dermal (LIVESEYSA, KERNDON DN.HOLLYOAK MA, et al.Transplanted acellular allograft dermalmatrix.Potential as a template for the reconstruction of viabledermis[J] .Transplantation, 1995,60:1-9.).The skin of Wainwright personnel selection make acellular dermal (WAINWRTIGHT DJ.Use of an acellular allograft deMal matrix (AlloDerm) in the management full-thickness bums[J] .Burns, 1995.21:243-248.).What have again subsequently that researcher will cultivate transplants and repairs the holostrome skin injury and succeed from the compound cell allosome dermal matrix that takes off of body surface chrotoplast diaphragm.U.S. LifeCell company makes acellular dermal matrix with this achievement commercialization with fresh people's cadaver skin, called after AlloDerm, and the approval of having passed through U.S. FDA has entered biomaterial for medical purpose market.2000, the Chen Si of this prince hospital of Hong Kong Chinese University's Weir is virtuous, LinBing Quan etc. cultivated human body from body epidermis cell was compounded in success 3 examples on the Integra substrate.Allosome corium is taken off that cell is handled to early starts such as domestic Chen Bi and clinical application research (Cui Zhengjun. Chen Bi, etc. in the Composite Skin research in the removal corium side of epithelial components allow and meaning [J]. Chinese shaping burns unit magazine, 1997.13 (1): 37-39.).1998, behind Sun Yonghua etc. report 37 routine fire victim III degree wound surface and the scar excision, carry out allosome acellular dermal+transplant, survive average out to (96.2 ± 3.4) % from body thin skin sheet, wound surface shrinks light, flat appearance, function is good, the satisfied (Sun Yonghua of clinical effectiveness, Li Chi, Wang Chunyuan, etc. take off cell allosome corium and the research and the application [J] of transplanting from body thin skin sheet. Chinese shaping burns unit magazine .1998,14 (4): 370-373.).Calendar year 2001, Feng Xiangsheng, Tan Jiaju, report xenogenesis pig acellular dermal matrix such as Pan Yingen with repair 23 routine fire victim III degree wound surface and scar excision wound surface from body surface skin cograft and as soft tissue depression patient's filler, the composite skin that survives is smooth, flexible, the same normal skin of color and long-term effect, smooth surface, flat appearance, touch soft, skin can have been pinched, no scar hyperplasia or slight scar hyperplasia, satisfied (the Feng Xiangsheng of clinical effectiveness, Pan Yingen, Tan Jiaju, etc. xenogenesis (pig) acellular dermal with from body surface skin cograft research [J]. Chinese shaping surgery magazine .2000.16 (1): 40-42.).Qidong, Jiangsu medical material institute has been released the xenogenesis acellular dermal matrix on this theoretical basis, and the approval that has obtained SFDA enters the covering of clinical practice in burn wound.Along with the application success of acellular dermal matrix in the burn field, the reparation of reconstruction of oral defect has also caused people's attention gradually, and has the part scholar to carry out clinical research with regard to acellular dermal matrix repairing reconstruction of oral defect.In April, 2003, method is red forever, Li Zhiren, Cai Xingwei use acellular dermal and repair Oral and Maxillofacial Surgery skin, mucomembranous defect and obtained satisfied effect that (method is red forever, Li Zhiren, etc. the application [J] of allosome soft tissue repair material in oromaxillo-facial region is damaged. modern stomatology magazine 2004.18 (1) 67-68.); August in the same year, stone is iced, the exposed maxillary of the high application organizes through engineering approaches of Song Qing oral mucosa reparation has been obtained good effect (stone is iced, Song Qing is high. and engineered oral mucosa is transplanted to repair and is exposed the research West China stomatology magazine 2003.8255-258. of hard palate to the maxillary growth development impact).The domestic market, the inferior company of Beijing name of the last ruler of the Xia Dynasty, Beijing Qingyuan Albert Biological Tissue Engineering Technology Co.ltd are applied to acellular dermal matrix in the product of skin injury reparation and reconstruction of oral defect reparation, and have all obtained the approval of Chinese SFDA.The allogeneic acellular dermal matrix, have following characteristics: 1. antigenicity is little; 2. hypotoxicity; 3. there are new vessels and fibroblast to grow into after implanting.But exist some shortcomings simultaneously: 1, raw material sources are narrow, and the misgivings of ethics aspect are arranged, and are difficult for popularizing; 2, cost an arm and a leg; 3, the danger of transmitted virus arranged.
Although the application of existing xenogenesis acellular dermal, but at present in the preparation method of acellular dermal matrix in order to reduce the immunogenicity of material, all use chemical fixatives in the method, as glutaraldehyde etc., this will change the natural structure of acellular dermal matrix greatly, and hinder acellular dermal matrix degraded in vivo.In addition, all do not have to consider how to kill the method for zoonosis pathogen such as bovine spongiform encephalopathy in the present acellular dermal matrix preparation method, this makes the clinical practice of acellular dermal matrix bear certain risk.
Animal derived medical apparatus and instruments kind is extensive.Animal tissue and its derivant are being better than no life entity material such as metal, plastics or textile aspect its characteristic.The quantity of animal tissue's raw material and kind also are various.These raw materials are as the main composition (as the heart of pig, cattle, Intestinum caprae seu ovis stitching thread, hemorrhage etc.) of medical apparatus and instruments, and product external structure or consolidate body (as heparin, gel, collagen etc.) perhaps is used for process of manufacture (as tallow).Present animal derived medical device product market prospect is wide.Infecting both domestic animals and human and particularly crazy cattle have also brought risk for animal derived medical apparatus and instruments.When finding that cattle suffers from bovine spongiform encephalopathy, the experts and scholars of states such as Britain, Germany, France have just begun to obtain the scientific research that possibility of infection has been carried out system to other animal susceptibility and with the mankind.Studies confirm that other animal that bovine spongiform encephalopathy can experimental infection has Mus, cattle, sheep, goat, pig, water Nyctereutes procyonoides or the like.Bovine spongiform encephalopathy all takes place in the gemsbok of Britain Safari Park and German zoo ostrich.Britain also have 69 cats eaten beef (bone) for made animal feed postoperative infection the report of bovine spongiform encephalopathy.To in by the end of February, 1997, Britain has had been found that 17 routine novel Ke Ya-syndrome people.Estimate that the people that Britain infects the refined syndrome of novel gram reaches 2,000,000 people, be 10-30 this sick incubation period, might occur restraining refined syndrome after 10 years and be very popular.At present, the ablation method of bovine spongiform encephalopathy pathogen is very limited.
Summary of the invention
The acellular dermal matrix that the purpose of this invention is to provide pathogen such as a kind of kill bacteria, virus and bovine spongiform encephalopathy.
Acellular dermal matrix provided by the present invention is successively with the product that obtains after sodium hydroxide solution and the detergent-treatment with mammal skin.
The concentration of described sodium hydroxide solution is 2-5N, and detergent can be tween 80, tween 20, TRITON-X100 or other chemical decontamination agent that mass percentage concentration is 1-5%.
Second purpose of the present invention provides a kind of preparation method of above-mentioned acellular dermal matrix.
The preparation method of above-mentioned acellular dermal matrix provided by the present invention may further comprise the steps:
1) peels off the mammal skin surface;
2) remove subcutaneous subsidiary composition;
3) clean skin;
4) skin was handled 10-60 minute with the 2-5N sodium hydroxide solution;
5) the new 2-5N sodium hydroxide solution of displacement was handled 10-60 minute;
6) repeating step 5) 1-3 time;
7) the skin mass percentage concentration that step 6) is handled through sodium hydroxide solution is that the detergent of 1-5% is handled;
8) spend pyrogen water skin is cleaned, obtain acellular dermal matrix.
In above-mentioned preparation method, the mammiferous selection that can be used for preparing acellular dermal matrix in the step 1) is diversified, comprises pig, cattle, sheep, horse or rabbit etc.
Step 2) the subcutaneous subsidiary composition in is meant the subcutaneous incidental fat of mammal, compositions such as muscle and hair.
Step 4)-6) concentration of sodium hydroxide solution is preferably 2N in, and the processing time is preferably 20-30 minute; The number of times of reprocessing is 1 to get final product in the step 6).
Used detergent can be tween 80, tween 20, TRITON-X100 or other chemical decontamination agent in the step 7), is preferably tween 80; The detergent-treatment time can be 10-60 minute, is preferably 30 minutes.
With the acellular dermal matrix of method for preparing can be stored in phosphate buffered solution (phosphatebuffered saline, PBS) in.
The invention provides a kind of acellular dermal matrix and preparation method thereof.In the preparation method of this acellular dermal matrix, adopting sodium hydroxide solution is fully to kill the pathogen of zoonosis such as bovine spongiform encephalopathy to its purpose of handling, can also remove the hereditary material-DNA of cell in the tissue effectively; The purpose of using detergent is the lipid material of removing in the cell membrane.The preparation method of acellular dermal matrix of the present invention has the following advantages: 1) acellular dermal matrix wide material sources, low production cost; 2) effectively go pathogen such as kill bacteria, virus and bovine spongiform encephalopathy, eliminated the zoonosis transmission danger; 3) effectively removed the antigenicity of material; 4) do not use fixative in the processing procedure, thereby prepared acellular dermal matrix kept natural structure, can be degraded, and guide tissue regeneration effectively.Experiment showed, that acellular dermal matrix of the present invention has the following advantages: 1) help repair cell and grow into wherein, induce fibroblast and newborn collagen to be arranged in the braiding shape, the structure of the approximate normal structure in healing back along its organizational structure; 2) histocompatibility is better, and easy and autologous tissue merges; 3) rejection is little; 4) can effectively improve tissue compliance in the wound healing process, the influence of pair cell and tissue compliance may have positive role to wound healing and the hyperplasia that reduces cicatrix.It is safe and effective as a kind of novel biomaterial, can be used for the skin trauma reparation, oral mucosa defect repair, hernia reparation, soft tissue repair such as urinary system tissue defect.The present invention has higher actual application value at medical domain.
The invention will be further described below in conjunction with specific embodiment.
Description of drawings
Fig. 1 is the photo of acellular dermal matrix after lyophilizing
Fig. 2 is the electron microscopic observation result of acellular dermal matrix internal structure
The fluorescence photo that Fig. 3 grows on acellular dermal matrix for human fibroblasts
Fig. 4 A is for carrying out the experimental implementation figure of subcutaneous rat embedding experiment to acellular dermal matrix
The observed result of the Histological section that Fig. 4 B is made into for taking a sample in the 1st week after implanting
The observed result of the Histological section that Fig. 4 C is made into for taking a sample in the 4th week after implanting
Fig. 5 is the PCR testing result of acellular dermal matrix DNA residual condition
Fig. 6 is for carrying out photo in the oral soft tissue shallow-layer defect repair operation process with acellular dermal matrix to upper right jaw malignant fibrohistiocytoma case
Fig. 7 is the photo in postoperative 2 all wound surface districts
Fig. 8 is the observed result of a postoperative experimental group wound healing situation the 1st, 2,3,4,6,12 Thursdays
The result of Fig. 9 under Electronic Speculum and light microscopic, the positive and negative of acellular dermal matrix of the present invention being observed
Figure 10 is light microscopic (200 times) observed result of postoperative 1 all tissue slices
Figure 11 is light microscopic (200 times) observed result of postoperative 2 all tissue slices
Figure 12 is light microscopic (200 times) observed result of postoperative 4 all tissue slices
Figure 13 is light microscopic (200 times) observed result of postoperative 6 all tissue slices
Figure 14 is light microscopic (200 times) observed result of postoperative 12 all tissue slices
Figure 15 is the skin histology compliance comparative result of four experimental group wound surface skin histologies
The specific embodiment
Method therefor is conventional method if no special instructions among the following embodiment, and described percent concentration is mass percentage concentration, and the primer is synthetic by Shanghai Bo Ya Bioisystech Co., Ltd.
The preparation of embodiment 1, acellular dermal matrix
With the sheep is the material source of preparation acellular dermal matrix, and concrete preparation process comprises the steps:
1) peels off the skin surface of sheep;
2) remove subcutaneous fat, subsidiary compositions such as muscle and hair;
3) with clear water skin is cleaned up;
4) with the 2N sodium hydroxide solution to skin treatment 40 minutes;
5) the new 2N sodium hydroxide solution of displacement was handled 40 minutes again;
6) step 5) is repeated once;
7) with 5% tween 80 to skin treatment 20 minutes;
8) spend pyrogen water the skin of handling through above-mentioned steps is fully cleaned, it is residual to remove chemical constituent, obtains acellular dermal matrix, and it is stored among the PBS.
The preparation of embodiment 2, acellular dermal matrix
With the cattle is the material source of preparation acellular dermal matrix, and concrete preparation process comprises the steps:
1) peels off the skin surface of cattle;
2) remove subcutaneous fat, subsidiary compositions such as muscle and hair;
3) with clear water skin is cleaned up;
4) with the 5N sodium hydroxide solution to skin treatment 30 minutes;
5) the new 5N sodium hydroxide solution of displacement was handled 30 minutes again;
6) step 5) is repeated secondary;
7) use 1%TRITON-X100, to skin treatment 30 minutes;
8) spend pyrogen water the skin of handling through above-mentioned steps is fully cleaned, it is residual to remove chemical constituent, obtains acellular dermal matrix, and it is stored among the PBS.
The constructed observation of embodiment 3, acellular dermal matrix and detection
One, the Electronic Speculum constructed observation of acellular dermal matrix
The photo of acellular dermal matrix after lyophilizing of embodiment 1 and embodiment 2 preparations as shown in Figure 1, again its internal structure is observed with Electronic Speculum, as shown in Figure 2, show that material space keeps the space of 20-100 micron size, prepared acellular dermal matrix is suitable for the growth of various types of cells.
Two, the biocompatibility of acellular dermal matrix detects
Human fibroblasts is inoculated on the acellular dermal matrix of embodiment 1 and embodiment 2 preparations, the cell number of inoculation is 10
6Individual, under 37 ℃ of conditions, cultivate, observer's growth of fibroblasts situation, the growing state of inoculation back cell as shown in Figure 3, human fibroblastic growth is vigorous, shows that prepared acellular dermal matrix has good cell compatibility.
Three, the immunogenicity of acellular dermal matrix detects
Acellular dermal matrix to embodiment 1 and embodiment 2 preparations carries out subcutaneous rat embedding experiment, whether can cause the reaction of animal immune originality to detect it, experimental implementation is shown in Fig. 4 A, respectively at implanting the back sampling of the 1st and the 4th week of back, being made into Histological section observes, the result shows that this acellular dermal matrix does not cause tangible immunogenic response shown in Fig. 4 B and Fig. 4 C.
Four, the detection at center is commented in the inspection of national medicine inspection office
The acellular dermal matrix of embodiment 1 and embodiment 2 preparations is delivered SFDA Jinan inspection center of national medicine inspection office, this acellular dermal matrix is detected, testing result is as follows:
1) aseptic qualified
2) bacterial endotoxin is qualified less than 0.5Eu/mL
3) hemolysis rate 0.8% (should be not more than 5%) is qualified
4) cell-cytotoxic reaction be not more than 1 grade qualified
5) no sensitization of skin reaction is qualified
6) no Intradermal irritant reaction is qualified
The above index that detects is all assert qualifiedly, and assert that this acellular dermal matrix has lower immunogenicity and degradability preferably.
Five, PCR detects the DNA residual condition of acellular dermal matrix
Detect with the method for PCR DNA residual condition the acellular dermal matrix of embodiment 1 and embodiment 2 preparations.From material, extract DNA and as template, the negative control template is a water, the positive control template is taken from the extractive DNA of animal skin, at primer 1:5 '-acgactcactatagggcttttttttttttgc-3 ' and primer 2: under the guiding of 5 '-acaatttcacacaggatacaa cgagg-3 ', carry out conventional pcr amplification, the PCR reaction condition is: earlier 95 ℃ 2 minutes; Again 94 ℃ 15 seconds, 49 ℃ 30 seconds, 72 ℃ 90 seconds, totally 5 circulations; Then 94 ℃ 15 seconds, 60 ℃ 30 seconds, 72 ℃ 90 seconds, totally 37 circulations; Last 72 ℃ 7 minutes.After reaction finishes, pcr amplification product is carried out agarose gel electrophoresis detect, (swimming lane M is DNA Marker to testing result as shown in Figure 5; The water belongs with yin contrast; Collagen 1 is the intermediate product in the acellular matrix preparation process; Collagen 2 is untreated skin histology; Collagen 3 is prepared acellular dermal matrix), show that all to detect DNA in intermediate product from the acellular matrix preparation process and the untreated skin histology residual, and it is residual not detect DNA from prepared acellular dermal matrix, proves that the preparation method of acellular dermal matrix of the present invention can thoroughly remove the cell residue composition.
The clinical trial of embodiment 4, oral soft tissue shallow-layer defect repair
Select upper right jaw malignant fibrohistiocytoma case 6 examples, outpatient service 4 examples, ward 2 examples.(specification: 5 * 5cm) carry out repairing test to the damaged case of above-mentioned oral soft tissue shallow-layer, and operation process as shown in Figure 6 with the acellular dermal matrix of embodiment 1 and embodiment 2 preparations.2 weeks of postoperative unpack, and observe the wound surface district and survive situation, and as shown in Figure 7, wound surface district color shows that near autologous tissue acellular dermal matrix of the present invention has repairing effect preferably to the case that has soft tissue or surface of bone to support.
Embodiment 5, skin repair experiment
The acellular dermal matrix of research embodiment 1 and embodiment 2 preparations whether have the improvement effect for wound healing after the operation of skin injury, to estimate its effectiveness and safety.Concrete experimental technique is: laboratory animal adopts SPF level male SD rat (Chinese Academy of Medical Sciences's Shanghai Experimental Animal Center), body weight 230-300g, the depilation of Rhizoma Atractylodis Macrocephalae barium sulfide the previous day, through 2.5% pentobarbital sodium (35mg/kg body weight) intraperitoneal injection of anesthesia, sterilization, animal is divided into 4 processed group at random: normal control group, simple wound group, positive controls and experimental group, every group each the time number of animals put mutually be 6.Above-mentioned four treated animals are carried out following processing: the normal control treated animal is not done special handling; Its excess-three treated animal cuts the holostrome skin of 2.5cm * 2.5cm in SD rat back center, simple wound is formed face and do not done the skin-grafting processing, lets alone normal healing after the wrapping; The positive controls wound surface is used commercial pig acellular dermal matrix (ADM) and autologous transplanting sword pachydermia; The acellular dermal matrix and the autologous transplanting sword pachydermia of experimental group wound surface Application Example 1 and embodiment 2 preparations are wrapped up with sterile gauze.Experimental result is as follows:
One, gross examination of skeletal muscle result
Observe the wound healing situation in the 1st, 2,3,4,6,12 weeks of postoperative, the result is (A among Fig. 8 is the normal control group) as shown in Figure 8.Simple wound group postoperative wound surface shrinks obviously, and wound surface is linear and (sees the B-D figure among Fig. 8, B: simple matched group postoperative 1 all wound surface after the epithelization; C: simple matched group postoperative 3 all wound surface; D: simple matched group postoperative 12 week healing wound surface).Positive controls postoperative 1 all time shift skin-grafting plate annesls, wound healing is good, transplants the skin graft well-grown in 4 weeks of postoperative and (sees E and G figure among Fig. 8, E: positive controls postoperative 1 all wound surface; G: positive controls postoperative 4 all wound surface), only do linear little otch and the experimental group embedded material is initial with scalpel, even be applied to easily form the dry and hard necrosis of ecchymosis skin graft behind the wound surface, part can local be survived preferably, and (see F and H figure among Fig. 8, F: experimental group (before improving) 1 week of postoperative is transplanted the dry and hard necrosis of skin graft; H: experimental group (improve before) the postoperative wound surface (local survival) preferably that heals during 4 weeks).Positive and negative to acellular dermal matrix used in the experimental group are observed under Electronic Speculum and light microscopic, and the result is (A: acellular dermal matrix reverse side surface (corium face) electromicroscopic photograph as shown in Figure 9; B: acellular dermal matrix front face surface (epidermis side) electromicroscopic photograph; C: structure under the acellular dermal matrix light microscopic (200 times)), acellular dermal matrix corium face crack is less, is unfavorable for tissue fluid infiltration and drain, may be the reason that causes the skin graft graft failure.In view of the above, operation plan is improved: when burrowing to acellular dermal matrix before improving is to make linear cut thereon with scalpel, consider this biofilm structure densification, matter is tough, then still be unfavorable for the infiltration and the drain of tissue fluid inadequately as kerf width, after the improvement, width with otch in to biomembrane punching process strengthens (about 0.5m), observe in the 1st, 4 weeks of postoperative again, transplant skin graft 1 all annesls after surgery, wound healing is good (sees I and J figure among Fig. 8, I: postoperative 1 all experimental grouies (improving the back) wound healing situation; J: postoperative 4 all experimental grouies (improving the back) wound healing situation), show that acellular dermal matrix helps transplanting the growth of skin graft after above-mentioned processing.Above-mentioned observed result shows that carrying out skin repair with acellular dermal matrix of the present invention can obtain healing effect preferably.
Two, om observation result
Experiment material is extracted in wound surface operation in the 1st, 2,3,4,6,12 weeks of postoperative from four experimental grouies, the size of leaving and taking histological specimen is about 0.5cm * 1cm, use 10% formalin fixed, make paraffin section, do HE dyeing, observe under optical microscope, the result is shown in Figure 10-14.
The skin tissue cell epimatrix of normal group matched group is arranged and is the braiding shape, therebetween the fibroblast of visible a small amount of engrain, as seen hair follicle and sweat gland structure (seeing A, E, I, M and Q figure among Figure 10-14).
The light microscopic of postoperative 1 all tissue slices (200 times) observed result is (A: the skin histology structure of normal control group as shown in figure 10; B: simple wound group, fibroblast is fusiformis, and cell and newborn collagen are parallel to skin surface; C: positive controls, visible inflammatory cell and the fibroblast of soaking among the ADM, visible obviously gap between ADM and autologous tissue; D: experimental group, the fibroblast and the newborn collagen of interstice's visible tissue structural arrangement traveling, cell is polygon.Acellular dermal matrix and autologous tissue do not merge, as seen obvious gap), postoperative 1-2 week, simple wound is formed face epithelization not as yet, wound surface is filled by a large amount of granulation tissuies, and inflammatory cell infiltration is obvious in the tissue, as seen the fibroblast of a large amount of dense arrangements, and cell is fusiformis, is parallel to skin surface with newborn collagen; A small amount of very thin collagen is arranged in the intercellular substance, obvious with the wound surface core especially; Blood capillary proliferation is obvious, and direction of travel is vertical with skin surface.All as seen, there are a large amount of outgrowth fibroblasts in interstice in the biomaterial that positive controls and experimental group are implanted, and fibroblast is polygon, rarely seen a spot of lymphocyte in the inflammatory cell of infiltration.Fibroblast is many in the experimental group biomaterial, and cell is light to be dyed.Positive controls fibroblast number is few, the cell engrain.The light microscopic of postoperative 2 all tissue slices (200 times) observed result is (E: the skin histology structure of normal control group as shown in figure 11; F: simple wound group, fibroblast is fusiformis, and quantity is many, and newborn collagen is very thin, is parallel to skin surface; G: positive controls, visible fibroblast among the ADM, amount is few, and ADM and autologous tissue easily differentiate; H: experimental group, fibroblast and newborn collagen are arranged with the biomembrane organizational structure, and cell is polygon.Acellular dermal matrix and autologous tissue merge, the original structure profile is not easily distinguishable), postoperative is during 1 week, the gap is big between experimental group biomaterial and autologous transplanting skin, merge as yet, postoperative during 2 weeks biomaterial with merge substantially from the body skin, do not have obvious gap and exist, and biomaterial is filled by fibroblast and newborn collagen, and the profile of raw material structure is not obvious.The biomaterial mirror of positive controls for puce, is differentiated with autologous transplanting skin and newborn collagen down easily mutually, and there be (seeing the S figure among Figure 14) in postoperative in still visible gap during 12 weeks and between the body skin.
Postoperative 3-4 week, simple wound group is finished epithelization, still visible more inflammatory cell infiltration in the tissue, fibroblast quantity obviously descends than postoperative is early stage, cell is spindle shape or flexing shape more, is arranged in parallel with skin surface, and cell space is big, light dying, newborn collagen is very thin, fine and close, be parallel to skin surface arranges.Experimental group and positive controls fibroblast quantity reduce, and are polygon, rare inflammatory cell.Experimental group biomaterial profile is not obvious, and this parts of fine extracellular matrix is the braiding shape to be arranged, but collagen is still more very thin.The light microscopic of postoperative 4 all tissue slices (200 times) observed result is (I: the skin histology structure of normal control group as shown in figure 12; J: simple wound group, fibroblast quantity reduces; K: positive controls, visible a small amount of fibroblast among the ADM, the ADM clear-cut is held by newborn fibrous tissue; L: experimental group, fibroblast and newborn collagen and biomembrane organizational structure are inseparable, and tissue is the braiding shape to be arranged, cell number is more).
The light microscopic of postoperative 6 all tissue slices (200 times) observed result is (M: the skin histology structure of normal control group as shown in figure 13; N: simple wound group, fibroblast quantity reduces, and collagen chap gradually is big; 0: positive controls, visible a small amount of engrain fibroblast among the ADM, the ADM clear-cut can be distinguished, and still visible gap between autologous tissue; P: experimental group, fibroblast quantity obviously reduces, organizational structure is the braiding shape and arranges similar normal structure structure), postoperative is during 6 weeks, and it is obviously less that each forms fibrocyte quantity, and experimental group wound surface fibroblast number is many than positive controls.
(Q: the skin histology structure of normal control group, tissue are the braiding shape and arrange the light microscopic of postoperative 12 all tissue slices (200 times) observed result as shown in figure 14; R: simple wound group, the fibroblast engrain, the collagen chap is big, the boundling shape; S: positive controls, visible a small amount of engrain fibroblast among the ADM, the ADM clear-cut can be distinguished, and still visible gap between autologous tissue; T: experimental group, fibroblast quantity obviously reduces, and organizational structure is the braiding shape and arranges, near the normal structure structure), 12 weeks of postoperative, simple wound form the chap of face portion collagen big, be the boundling shape, arrange and tend to normal skin tissue, visible a spot of fibroblast gradually; Experimental group wound surface collagen is thick, the boundling shape, arrange and to be the braiding shape, and the fibroblast of visible a small amount of engrain is arranged near the collagen of normal skin tissue.The biomaterial clear-cut that positive controls is implanted.The om observation result of above-mentioned tissue slice shows with the used ADM of positive controls and compares that acellular dermal matrix of the present invention more helps growing into of repair cell, easily with autologous tissue's fusion.
Have rejection in various degree after allosome or foreign material are implanted, rejection is unfavorable for the healing of wound surface excessively by force.Under observation find, compare with ADM, behind acellular dermal matrix implantation rat wound surface of the present invention, also do not see that the especially phenomenon of lymphocytic infiltration of a large amount of inflammatory cells appears in wound tissue, the rejection that shows generation is less, and its rejection degree is little to the negative effect of wound healing.
Three, the detection of wound surface skin histology compliance
In postoperative the 1st, 2,3,4,6,12 weeks were extracted experiment material from the wound surface operation of four experimental grouies, detect the tissue compliance of four experimental group wound surface skin histologies, assay method is: laterally cut wide 0.5cm from starting from the wound surface middle part in 2 weeks of postoperative, two ends (start from wound surface around normal skin) are apart from one of the skin graft of each 2.5cm of edge of wound, cut off subcutaneous tissue, measure the length of specimen wound surface part, thickness and skin graft width (are partly got three point measurements in wound surface, get its meansigma methods), with INSTRON biological mechanics determining instrument (INSTRON 5542-C8609), with loading 20N, 0.5mm/min constant speed stretching skin graft, stretching distance is 10% Δ L (length of skin graft wound surface part), measures the corresponding loading of four experimental group wound surface skin histologies.The skin histology compliance is the deformability of tissue, represents (compliance and loading are inverse ratio) with the inverse of the loading of unit volume, unit displacement, and computing formula is as follows
The data statistics result of the skin histology compliance of four experimental group wound surface skin histologies is as shown in table 1, comparative result as shown in figure 15,2 weeks of postoperative are the tissue compliance increase of wound group merely, is higher than normal group, and both compare has significant difference (P＜0.05); The tissue compliance of the simple wound group in 2 week backs reduces, and significantly is lower than normal group (P＜0.05); Postoperative 2 during week the tissue compliance of experimental group and positive controls significantly be lower than simple wound group, be significantly higher than simple wound group (P＜0.05) postoperative 3-12 week, after showing the rat skin tissue defect, as the healing of giving free rein to, the wound tissue compliance obviously descends, and cograft (biomaterial+self-skin transplant) can obviously improve the tissue compliance of wound surface behind the rat skin tissue defect, existing studies confirm that, the decline of tissue compliance can impel Fibroblast Function active, is one of key factor of cicatrix hyperplasia.Cograft will help alleviating the hyperplasia of cicatrix to the improvement of tissue compliance.The tissue compliance of experimental group significantly is lower than positive controls (P＜0.05) after surgery 3-4 week, all the other the time difference of putting mutually that there are no significant, this result is consistent with fibroblast quantity observed result more, that function is active in last this time period tissue of histology, show at wound healing early stage, acellular dermal matrix of the present invention is slightly poor than ADM to the improvement of tissue compliance, but its long-term effect is no significant difference between the two, and therefore acellular dermal matrix of the present invention also can effectively improve the tissue compliance of wound surface.In addition, wound healing is early stage, simple wound is formed the covering weave compliance and is significantly higher than other group, may be early stage because of wound healing, wound surface is filled by a large amount of new granulation tissues, main in the new granulation tissue at this moment based on a large amount of cell component, and extracellular matrix is less relatively, arranges to loosen, so its compliance is higher on the contrary, healing along with wound surface, finishing of epithelization, a large amount of depositions of extracellular matrix, it is fine and close that the arrangement of extracellular matrix becomes, non-deformability improves, so its compliance obviously descends.
Table 1 different disposal is formed the data analysis result of surface skin tissue compliance
(n=4-6)
| 2 weeks of postoperative | 3 weeks of postoperative | 4 weeks of postoperative | 6 weeks of postoperative | 12 weeks of postoperative | |
| Normal group | 4.36±0.00215 | 2.27±0.00062 | 2.84±0.0004 | 2.68±0.00087 | 6.12±0.00184 |
| Simple wound group | 1.83±0.00125 △ | 6.96±0.01266 △ | 5.59±0.00064 △ | 16.49±0.03039 △ | 15.74±0.03008 △ |
| Positive controls | 3.10±0.00139 | 1.90±0.00004 | 2.90±0.00125 | 5.52±0.01625 | 5.35±0.00065 |
| Experimental group | 2.54±0.00066 | 2.98±0.0002 ▲ | 5.66±0.00058 ▲ | 4.56±0.0013 | 5.72±0.00169 |
Annotate:
#Each data all multiply by 10
3 △Normal group, positive controls, experimental group and simple wound group be p＜0.05 relatively;
▲Experimental group and positive controls be P＜0.05 relatively.
Claims (8)
1. an acellular dermal matrix is successively with the product that obtains after sodium hydroxide solution and the detergent-treatment with mammal skin; This processing procedure may further comprise the steps:
1) peels off the mammal skin surface;
2) remove subcutaneous subsidiary composition;
3) clean skin;
4) skin was handled 10-60 minute with the 2-5N sodium hydroxide solution;
5) the new 2-5N sodium hydroxide solution of displacement was handled 10-60 minute;
6) repeating step 5) 1-3 time;
7) the skin mass percentage concentration that step 6) is handled through sodium hydroxide solution is that the detergent of 1-5% is handled;
8) spend pyrogen water skin is cleaned, obtain acellular dermal matrix.
2. acellular dermal matrix according to claim 1 is characterized in that: described detergent is that mass percentage concentration is tween 80, tween 20 or the TRITON-X100 of 1-5%.
3. acellular dermal matrix according to claim 1 is characterized in that: the mammal that is used to prepare acellular dermal matrix in the described step 1) is pig, cattle, sheep, horse or rabbit.
4. acellular dermal matrix according to claim 1 is characterized in that: the concentration of sodium hydroxide solution is 2N described step 4)-6), and the processing time is 20-30 minute.
5. according to claim 1 or 4 described acellular dermal matrixes, it is characterized in that: the number of times of reprocessing is 1 in the described step 6).
6. acellular dermal matrix according to claim 1 is characterized in that: the detergent in the described step 7) is tween 80, tween 20 or TRITON-X100.
7. according to claim 1 or 6 described acellular dermal matrixes, it is characterized in that: the detergent-treatment time is 10-60 minute in the described step 7).
8. acellular dermal matrix according to claim 7 is characterized in that: described detergent is a tween 80, and the processing time is 30 minutes.
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